Changes in the mitochondrial network during ectromelia virus infection of permissive L929 cells

Mitochondria are extremely important organelles in the life of a cell. Recent studies indicate that mitochondria also play a fundamental role in the cellular innate immune mechanisms against viral infections. Moreover, mitochondria are able to alter their shape continuously through fusion and fission. These tightly regulated processes are activated or inhibited under physiological or pathological (e.g. viral infection) conditions to help restore homeostasis. However, many types of viruses, such as orthopoxviruses, have developed various strategies to evade the mitochondrial-mediated antiviral innate immune responses. Moreover, orthopoxviruses exploit the mitochondria for their survival. Such viral activity has been reported during vaccinia virus (VACV) infection. Our study shows that the Moscow strain of ectromelia virus (ECTV-MOS), an orthopoxvirus, alters the mitochondrial network in permissive L929 cells. Upon infection, the branching structure of the mitochondrial network collapses and becomes disorganized followed by destruction of mitochondrial tubules during the late stage of infection. Small, discrete mitochondria co-localize with progeny virions, close to the cell membrane. Furthermore, clustering of mitochondria is observed around viral factories, particularly between the nucleus and viroplasm. Our findings suggest that ECTV-MOS modulates mitochondrial cellular distribution during later stages of the replication cycle, probably enabling viral replication and/or assembly as well as transport of progeny virions inside the cell. However, this requires further investigation

Ectromelia Virus Affects Mitochondrial Network Morphology, Distribution, and Physiology in Murine Fibroblasts and Macrophage Cell Line

Mitochondria are multifunctional organelles that participate in numerous processes in response to viral infection, but they are also a target for viruses. The aim of this study was to define subcellular events leading to alterations in mitochondrial morphology and function during infection with ectromelia virus (ECTV). We used two different cell lines and a combination of immunofluorescence techniques, confocal and electron microscopy, and flow cytometry to address subcellular changes following infection. Early in infection of L929 fibroblasts and RAW 264.7 macrophages, mitochondria gathered around viral factories. Later, the mitochondrial network became fragmented, forming punctate mitochondria that co-localized with the progeny virions. ECTV-co-localized mitochondria associated with the cytoskeleton components. Mitochondrial membrane potential, mitochondrial fission–fusion, mitochondrial mass, and generation of reactive oxygen species (ROS) were severely altered later in ECTV infection leading to damage of mitochondria. These results suggest an important role of mitochondria in supplying energy for virus replication and morphogenesis. Presumably, mitochondria participate in transport of viral particles inside and outside of the cell and/or they are a source of membranes for viral envelope formation. We speculate that the observed changes in the mitochondrial network organization and physiology in ECTV-infected cells provide suitable conditions for viral replication and morphogenesis

ECTV infection does not trigger phenotypic maturation and inhibits LPS-induced maturation of GM-BM.

<p>Mock-, uvi-ECTV- or ECTV-infected GM-BM were left untreated or were treated with LPS for 24 h. Representative histograms showing the major histocompatibility complex (MHC) (A) or co-stimulatory (B) molecules expression on GM-BM. Numbers represent the MFI value and/or the percentage of positive cells for a given marker. Graphs show mean ± SD of MFI and/or percentage for indicated marker from at least three independent experiments (paired Student’s <i>t</i>-test; ^*<i>P</i>< 0.05, ^**<i>P</i>< 0.01). Statistical comparisons were between mock- or uvi-ECTV-treated DCs and ECTV-exposed DCs and between LPS- or uvi-ECTV+LPS-treated DCs and ECTV + LPS-exposed DCs. IsCon–isotype control.</p

Functional paralysis of GM-CSF–derived bone marrow cells productively infected with ectromelia virus

<div><p>Ectromelia virus (ECTV) is an orthopoxvirus responsible for mousepox, a lethal disease of certain strains of mice that is similar to smallpox in humans, caused by variola virus (VARV). ECTV, similar to VARV, exhibits a narrow host range and has co-evolved with its natural host. Consequently, ECTV employs sophisticated and host-specific strategies to control the immune cells that are important for induction of antiviral immune response. In the present study we investigated the influence of ECTV infection on immune functions of murine GM-CSF–derived bone marrow cells (GM-BM), comprised of conventional dendritic cells (cDCs) and macrophages. Our results showed for the first time that ECTV is able to replicate productively in GM-BM and severely impaired their innate and adaptive immune functions. Infected GM-BM exhibited dramatic changes in morphology and increased apoptosis during the late stages of infection. Moreover, GM-BM cells were unable to uptake and process antigen, reach full maturity and mount a proinflammatory response. Inhibition of cytokine/chemokine response may result from the alteration of nuclear translocation of NF-κB, IRF3 and IRF7 transcription factors and down-regulation of many genes involved in TLR, RLR, NLR and type I IFN signaling pathways. Consequently, GM-BM show inability to stimulate proliferation of purified allogeneic CD4⁺ T cells in a primary mixed leukocyte reaction (MLR). Taken together, our data clearly indicate that ECTV induces immunosuppressive mechanisms in GM-BM leading to their functional paralysis, thus compromising their ability to initiate downstream T-cell activation events.</p></div

ECTV infection inhibits nuclear translocation of NF-κB, IRF3 and IRF7 in GM-BM.

<p>Indirect immunofluorescence analysis of nuclear translocation of NF-κB (A), IRF3 (B) and IRF7 (C) in mock-, uvi-ECTV- or ECTV-infected GM-BM untreated or treated with LPS for 24 h. Left panels show images of red fluorescence channel for the indicated transcription factor, right panel show images of merge fluorescence channels for the indicated transcription factor (red), viral antigen (green), and nuclear and viral DNA (blue). Arrowheads show viral factories (A, C) or vacuoles (B) in infected cells. Scale bars = 10 μm. Nuclear [N]: cytoplasmic [C] ratios of NF-κB (D), IRF3 (E) and IRF7 (F) were determined at single cell level by measuring of fluorescence signal intensities within the nucleus (stained with Hoechst 33342) and the cytosol. Analysis was performed on 50 cells/condition and experiment (from three independent experiments). Black lines indicate the mean values of each data set (Student’s <i>t</i>-test;^**<i>P</i>< 0.01).</p

ECTV infection modulates the expression of genes engaged in the innate antiviral immune response.

<p>Log2 fold change of mRNA expression for genes associated with Toll-like receptor (TLR) (A), RIG I-like receptor (RLR) (B), IFN type I (C) and NOD-like receptor (NLR) (D) signaling. Average threshold cycle (C_T) values from PCR reactions were normalized against the average C_T values for the endogenous control <i>Atg12</i> from the same cDNA sample and shown as 2^(-ΔΔCT) were ΔC_T = C_{T gene}− C_{T atg12}. Graph columns represent the mean values of log2 fold change in mRNA expression from two independent experiments (Student’s <i>t</i>-test; ^*<i>P</i>< 0.05, ^**<i>P</i>< 0.01).</p

ECTV induces morphological changes and apoptosis in infected GM-BM.

<p>(A) Morphological changes of GM-BM infected with ECTV. GM-BM were cultured in medium only (mock), medium containing UV-inactivated ECTV (uvi-ECTV) or live-ECTV (ECTV) and were stimulated with or without LPS (1 μg/ml) for 24 h. Left panels show representative images of May-Grünwald-Giemsa stained GM-BM. Right panels demonstrate representative scanning electron microscopy micrographs of GM-BM. Scale bars = 5 μm. (B) The mean percentage of early and late apoptotic cells in GM-BM at 4, 12 and 24 hpi. Error bars represent ± SD from three independent experiments (Student’s <i>t</i>-test; ^*<i>P</i>< 0.05, ^**<i>P</i>< 0.01).</p

Kinetics of ECTV replication in GM-BM.

<p>GM-BM were cultured in medium only (mock), medium containing UV-inactivated ECTV (uvi-ECTV) or live-ECTV (ECTV) for 4, 12 and/or 24 h. In some experiments cells were left untreated or were additionally treated with LPS (1 μg/ml). (A) Representative images of mock–, uvi-ECTV–and ECTV–treated GM-BM at 4 and 24 hpi stained with Hoechst 33342 (blue fluorescence) and pAbs anti-ECTV (green fluorescence). The magnified images are of the boxed regions. Arrows indicate viral particles; arrowheads show viral factories. Scale bars = 10 μm. (B) Representative histograms showing the percentage of ECTV⁺ cells at 4, 12 and 24 hpi of GM-BM. Numbers represent the percentage of ECTV⁺ cells. (C) The mean percentage of ECTV⁺ cells during infection in GM-BM. Error bars represent ± SD from three independent experiments.(D) Scanning electron microscopy micrograph of GM-BM surface at 24 hpi. The magnified images are of the boxed regions. Arrows indicate viral particles. Scale bars = 2.5 μm. IsCon–isotype control.</p